Hepatitis C virus (HCV) infection is most often clinically inapparent and rarely associated with symptoms of acute hepatitis. It is therefore difficult to study immune correlates of viral clearance during the very early phase of HCV infection in humans. The chimpanzee is the only animal model that is susceptible to HCV infection and suitable to study cellular immune responses at the site of infection, the liver. To define immunological correlates of recovery from hepatitis C in this model, we prospectively investigated the role of HCV-specific T cells in the liver of 5 chimpanzees during acute HCV infection. Although CD8+ T cells constituted the largest population of intrahepatic T cells during acute hepatitis, ex vivo flow cytometry demonstrated that significantly more CD4+ T cells than CD8+ T cells were activated. Moreover, we identified a subpopulation of CD4+ T cells that coexpressed CD8a/b and displayed the highest level of activation as measured by direct ex vivo analysis of DR expression. In addition, 88% (7/8) pure CD4+CD8+ T cell lines but only 59% (13/22) single CD4+ T cell lines were HCV specific. These CD4+CD8+ double positive cells resembled CD4+ single positive cells in their MHC class II restriction, proliferation and in their lack of cytotoxicity. In contrast to CD4+ T cells, however, they displayed a specific cytokine profile with constitutive IFN-g expression and an HCV-specific IL-4 and IFN-g recall response. This cytokine profile was especially dominant during the memory T cell response of the recovered chimpanzee when it cleared the HCV rechallenge inoculum without evidence of liver disease. In summary, the data suggest that a highly activated, noncytopathic subpopulation of CD4+ T cells contributed to HCV clearance and that their effector functions was mediated by a specific cytokine profile. To determine whether those cellular immune responses were protective, we rechallenged three HCV recovered, HCV envelope antibody negative chimpanzees with increasing doses of homologous HCV (1a) and one animal subsequently with heterologous HCV (1b). Components and kinetics of the HCV-specific immune response in the blood and liver were analyzed with regard to frequency and effector function of HCV-specific CD4+ and CD8+ T cells by tetramer staining, cytokine enzyme linked immunospot assays, proliferation and cytotoxicity assays and as related to the clinical and virological outcome. CD4+ memory T cells represented the most sensitive components of the HCV-specific immune response, because proliferative and IFNg CD4+ T cell recall responses were observed even after challenge with low titer inocula that caused neither detectable viremia nor CD8+ T cell responses. When viremia occurred after rechallenge with higher dose inocula, all chimpanzees controlled viral load at 2-4 log lower levels than in the previous infection. Viral clearance was achieved within 2-5 weeks without significant liver enzyme elevation. During this phase, HCV-specific CD4+ T cells responses preceded CD8+ T cell responses. Specifically, CD4+ T cells proliferated and produced IFN-g immediately at the onset of viremia. HCV-specific CD4+ T cells were also the predominant T cell population expanded from the liver during rechallenge and were targeted against all viral nonstructural proteins needed to be maintained for several months to completely eradicate the virus. To analyze CD8+ T cells responses, we have sequenced and identified common chimpanzee MHC, also called Patr-alleles and identified the panel of HCV peptides presented by those alleles and recognized by HCV-specific T cells. In contrast to CD4+ T cells that were detectable directly at the onset of viremia, HCV-specific, tetramer+ CD8+ T cells were functionally silent at that time and required a functional switch from proliferating CCR7+ central memory cells to cytokine producing CCR7- effector memory cells and started to produce IFN-g only after the virus was cleared. In summary, HCV-specific memory T cells of recovered chimpanzees responded vigorously to HCV rechallenge resulting in reduced viremia, earlier viral clearance and significantly less liver disease than in primary HCV infection. This outcome was associated with an adaptive cellular immune response to HCV in the absence of any detectable antibodies to HCV structural proteins.